Glycosidase activities in Chinese hamster ovary cell lysate and cell

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Glycosidase Activities in Chinese Hamster Ovary Cell Lysate and Cell Culture Supernatant Michael J. Gramer and Charles F. Goochee* Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025

T o probe the potential for extracellular degradation of glycoprotein oligosaccharides in conjunction with Chinese hamster ovary (CHO) cell culture, an initial characterization of several CHO cell glycosidases was performed using 4-methylumbelliferyl substrates. CHO cell lysates contained sialidase, 8-galactosidase, P-hexosaminidase, and fucosidase activities with pH optimums near 5.5, 4, 6, and 6.5, respectively. These glycosidase activities were also present in cell-free supernatant samples from commercial CHO cell cultures. The sialidase activity was further characterized. In contrast t o previous reports concerning mammalian sialidases, the sialidase activity in CHO cell lysate retained considerable activity a t pH 7 and was very stable, with a half-life of 57 h a t 37 OC. Both the K, and V,, of CHO lysate sialidase for 2’-(4-methylumbelliferyl)-cu-~-Nacetylneuraminic acid (4MU-NeuAc)varied with pH, and this activity was competitively inhibited by 2,3-dehydro-2-deoxy-N-acetylneuraminicacid and by free N-acetylneuraminic acid. The kinetic characteristics and pH-activity profiles of the CHO cell lysate and cell culture supernatant sialidase activities were essentially identical, and both released sialic acid from the glycoprotein fetuin at pH 7.5. These results suggest that the oligosaccharides of glycoproteins secreted by CHO cells can potentially be modified extracellularly by sialidase under culture conditions which promote the release and extracellular accumulation of this enzyme.

Introduction Glycoproteins have many important pharmaceutical applications. For example, glycoproteins are being used for clinical treatment of chronic renal failure (erythropoietin), cancer (antibodies), heart disease (tissue plasminogen activator), Gaucher’s disease (glucocerebrosidase), hemophilia (factor VIII), and cystic fibrosis (deoxyribonuclease). Most cell surface receptors are glycoproteins. Therefore, the use of purified cell surface receptors as targets for drug discovery defines another important pharmaceutical application of glycoproteins. Heterogeneity of oligosaccharide structure is typically observed at each N-linked or O-linked glycosylation site, leading to the concept of “g1ycoforms”-that is, a set of moleculeswith identical amino acid sequences but distinct oligosaccharidestructures (Rademacher et al., 1988).Much of the heterogeneity that has been observed among glycoforms of Chinese hamster ovary (CHO) produced glycoproteins such as tissue plasminogen activator (t-PA) (Spellman et al., 1989;Parekhet al., 1989),erythropoietin (EPO) (Sasaki et al., 1988), CD4 (Spellman et al., 1991; Yuen et al., 1990),and interferon-61 (Kagawa et al., 1988) is due to variabilityin the presence of sialic acid and fucose. Sialic acid is a terminal sugar and the only charged monosaccharide present on glycoproteinoligosaccharides. The presence or absence of sialic acid can affect many glycoprotein properties, including specific activity (Goldwasser et al., 1974;Briggs et al., 1974; Chavin et al., 1984; Tsuda et al., 1990; Yan et al., 1990; Smith et al., 1990; Takeuchi et al., 1990), antigenicity (Schauer, 19881, resistance to protease attack (Goldwasser et al., 1974), and resistance to thermal denaturation (Goldwasser et al., 1974; Tsuda, 1990). Glycoproteins lacking sialic acid

* Author to whom correspondence should be addressed. 8756-7938/93/3009-0366$04.00/0

are frequently much less soluble than their sialylated counterparts, a factor that can lead to potential problems in the purification and formulation of glycoprotein therapeutics. The absence of sialic acid on glycoprotein oligosaccharides results in rapid in vivo clearance by the asialoglycoprotein receptor (Weiss and Ashwell, 1989).As a result, EPO (Fukuda et al., 1989) or granulocytemacrophage colony-stimulatingfactor (GM-CSF) (mammalian-produced) (Donahue et al., 1986)lacking terminal sialic acid is ineffective in vivo. Thus, the presence of terminal sialic acid is a desirable feature for human therapeutic proteins requiring a long circulatory half-life. Given these considerations,it is important to understand how bioprocess conditions affect the sialic acid content of glycoprotein oligosaccharides. Such information is important during process development to maximize the titer of the desired glycoforms and to assure that the final process routinely yields the same distribution of glycoforms that was present in the product that went through clinical trials. Variability in sialic acid content can arise from stochastic events in oligosaccharidebiosynthesis. These biosynthetic events are potentially influenced by the cell culture environment (Goocheeand Monica, 1990). There has been no previous study exploring the possibilitythat additional heterogeneity of glycoprotein oligosaccharidesmight arise due to extracellular glycosidases endogenous to the host cell line. For extracellular modification to be an issue, glycosidases must be synthesized and released into the extracellular environment where they must be stable and active toward glycoprotein oligosaccharides. For modification during cellculture, the glycosidasesmust be active near pH 7. Glycosidase activity at lower or high pH might be important during subsequent purification steps. Mammalian cells possess endogenous glycosidases (Conzelmann and Sandhoff, 19871, raising the possibility for

0 1993 American Chemical Society and American InstRute of Chemical Engineers

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glycosidase release upon cell lysis during cell culture. from the dish and each other. Cells were removed from the dish with a cell lifter (Costar),and the cell suspension Another potential means for accumulation of extracellular was transferred to a centrifuge tube. At this time, some glycosidase activity is secretion due to intracellular misof the suspension was removed for cell counts, while the sorting events cawed, for example, by the accumulation remainder was centrifuged at l00g for 10 min. The PBS of extracellular ammonium ion (Hasilik and Neufeld, was aspirated, and the cells were resuspended in cold water 1980). Fucosidase, 8-galactosidase, and 8-hexosaminidase for osmotic lysis at a concentration of 1 X 107 cells/mL activities can accumulate in the extracellular medium of CHO cells after overnight incubation, demonstrating that (-3.3 mg/mL). Complete disruption was accomplished glycosidases are synthesized by CHO cells and that they by passing the cells through a 22-gauge needle five times. Lysate was either used fresh for assays or frozen for future may be released under cell culture conditions (Robbins, use. All operations above were performed under sterile 1979; Hall et al., 1986). The relevance of these data to conditions. Cell counts were performed with a Coulter extracellular modification of glycoproteinoligosaccharides Multisizer (Coulter Scientific Products) after sufficient is unclear since the activities were measured at acidic pH pipeting to break up clumps as determined by the cell size with artificial substrates. distribution profile. Protein concentrations were deterThe current literature data for mammalian sialidase mined with the BCA kit from Pierce using bovine serum activities do not strongly support the probability for albumen as a standard. Mycoplasma tests using the extracellular hydrolysis of sialic acid from glycoprotein Mycoplasma Rapid Detection System (GenProbe) were oligosaccharides in conjunction with cell culture. Mamnegative. malian sialidase enzymes have been reported to be T-Flask CHO Cell Culture Supernatant. DHFRextremely unstable in cell lysates (Conzelmann and WB1 CHO cells were plated and grown as described above Sandhoff, 1987;Potier et al., 1979a;Den Tandt and Leroy, in a T-150 tissue culture flask containing 30 mL of medium. 1980; Nguyen Hong et al., 1980; Hiraiwa et al., 1987), and After 2 days, the medium was harvested, centrifuged at most have acidic pH optimums with little activity near lOOg to remove any unattached cells, and concentrated by pH 7 (Warner and O'Brien, 1979; Spaltro and Alhadeff, a factor of 17 using a centrifugal ultrafilter (Amicon 1984;Schauer and Wember, 1984;Sato, 1989;Potier et al., centriprep) with a loo00 MW cutoff. The cells were 1979b). Sialidaseactivity has recently been demonstrated removed from the flask by trypsinization and counted using in CHO detergent extracts at pH 6.5 using the artificial substrate 2 ' - ( 4 - m e t h y l u m b e l l l ) - ~ - ~ ~ - a c e t y l n e u -the Coulter Multisizer. Cell viability was estimated using trypan blue exclusion. ic acid (4MU-NeuAc) (Potvin and Stanley, 1991). HowChiron CHO Cell Culture Supernatant. Frozen (3-5 ever, this activity was not further characterized. months at -80 "C) cell-free supernatant samples were In this study, we explorethe presence of four glycosidases provided by Chiron Corp. (Emeryville, CA) from two CHO in CHO cell lysate and CHO cell supernatant as a starting perfusion cultures. Recombinant CHO cells were proppoint for evaluating the potential for extracellular hyagated in a 40-L Delco bioreactor. The cells were perfused drolysis of glycoproteinoligosaccharides. The pH-activity with media consisting of DME/F12 containing insulin (5 profiles of sialidase, 8-gaactosidase, 8-hexosaminidase,and pg/mL), transferrin (1pg/mL), glutamine (380 pg/mL), fucosidase in CHO cell lysate are examined. The presence 0.3% fetal calf serum (heat-inactivated at 56 "C for 30 of these glycosidases is also evaluated in CHO cell culture min), and 1.45 mL/L of Excyte VLE (Miles Diagnostics). supernatants provided by two biotechnology companies. The perfusion rate was modulated from approximately The study concludes with an examination of the stability 0.1 to 0.5 (v/v)per day on the basis of the estimated glucose and kinetic characteristics of the sialidase activity in CHO consumption rate. The pH was maintained in the range cell lysate and CHO cell culture supernatant. of pH 6.7-6.9, and the cell density was approximately 2 X 106 cells/mL. The parental cell line used by Chiron was Materials and Methods the DG44 DHFR- CHO cell line (Urlaub et al., 1986).The General. All materials were from Sigma unless othrecombinant cells were grown in free suspension, and they erwise stated. Water was 18MQ from a Milli-Q five-bowl existed as single cells or as aggregates up to 100 cells. The system (Millipore). cells were secreting a low level (1-5 pg/mL) of recombinant glycoprotein. CHO Cell Lysate. The cell line used in this study was the WB1 DHFR- CHO cell line (DHFR, dihydrofolate Sialidase from Genentech CHO Cell Culture Sureductase) obtained from Genentech (South San Francisco, pernatant. A WB1 CHO cell line secreting a recombinant glycoprotein product was propagated in batch culture at CA). These cells are a subclone of the DHFR- CHO-K1DUX-B11 cell line (Simonsen and Levinson, 1983). The Genentech using a serum-free DME/FlB-based medium. WB1 DHFR- CHO cell line was grown in a humidified Concentrated, cell-free CHO cell culture supernatant was incubator at 37 "C with 5% COZin T-flasks (Corning). loaded onto an ion-exchange column, which strongly Medium (Sigma) consisted of DME/F12 with glutamine retained the majority of glycoprotein product. The and HEPES, supplemented with additional 2 mM sialidase was weakly retained, and approximately 80% of glutamine (Gibco), 5% heat-inactivated (45 min at 56 "C) the sialidase activity was recovered in the column wash. fetal bovine serum (Hyclone), antibiotics (100 unita/mL This fraction was supplied by Genentech and was subpenicillin G, 100 pg/mL streptomycin, and 25 pg/mL sequently concentrated for sialidase assays in this study. amphotericin B) (Gibco), and GHT (final concentrations: Artificial Substrate Glycosidase Assays. The ar50 pg/mL glycine, 10 pg/mL hypoxanthine, 10 pg/mL tificial substrate 4MU-NeuAc was used to assay for thymidine) (Kaufman, 1990). For lysate enzyme assays, sialidase activity modified from that previously described confluent cells (-5 X 106 cells/cm2) were trypsinized and (Potier et al., 1979b). The standard assay for lysate passaged 1:4 into 100-mm tissue culture dishes (Costar) sialidase activity contained 0.1 M sodium acetate/HCl (pH containing 15 mL of medium. Cells were harvested 1-2 3-6) or potassium phosphate/NaOH (pH 6-8) buffer (10 days later by first washing twice with 10 mL of cold pL of 1M stock), 1mM 4MU-NeuAc (25 pL from a 4 mM phosphate-buffered saline (PBS: 8 g/L NaC1,0.2 g/L KC1, aliquot), and the lysate of -0.25 million cells (25 pL of 1.13 g/L Na2HPO4,and 0.2 g/L KHzP04, pH 7.5). A third lysate) in a total volume of 100 pL. The 100-pL samples 10-mL aliquot of cold PBS was allowed to sit on the cells in plastic 1.5-mL tubes were placed at 37 "C in a water for 10 min at 4 "C, which aided in the loosening of cells bath shaker for 30 min, and the reaction was stopped with

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0.9 mL of 0.2 M glycine/NaOH buffer (pH 10.4). The samples were centrifuged at 12000g for 15 min in a microcentrifuge (Eppendorf) to remove large cellular components and diluted 1OX further into HPLC sample bottles (50-pL sample into 450 pL of glycine buffer). An HP 1090Series I1LC coupled to an H P 1046Aflow-through fluorescence detector (Hewlett-Packard) was used for automated sample injection and analysis (no column). The parameters were as follows: injection volume, 25 pL; eluant, 0.5 mL/min water; excitation, 362; emission, 448; photomultiplier gain, 10-13; cutoff filter, 370 nm; slit widths, 4 X 4 , l X 1,and 1 X 1mm; lamp setting, 1; run time, 0.5 min. The fluorescence was integrated vs time and compared to standard 4-methylumbelliferyldilutions. Controls included lysate without substrate, substrate without lysate, and blanks containing only the buffers. The other glycosidases were assayed similarly using appropriate substrates: 8-galactosidase, 4-methylumbelliferyl-8-D-galaCtOSe;&hexosaminidase, 4-methylumbelliferyl-8-D-N-acetylglucosamine;fucosidase, 4-methylumbelliferyl-a-L-fucose. Cell culture supernatant samples were assayed similarly, with supernatant replacing the added cell lysate. GlycoproteinSialidase Assays. Release of sialic acid from a glycoprotein substrate by CHO lysate sialidase was measured using fetuin (Gibco) as the substrate. The contents of glycoprotein sialidase assays were as above for the artificial substrate, except that glycoprotein-bound sialic acid was present instead of the artificial substrate. Released sialic acid was quantified by the thiobarbituric acid assay as previouslydescribed (Aminoff,1961);reagent volumes were reduced proportionally to accommodate a 100-pL sample volume, and the amount of acid butanol was reduced further to 400 pL to increase sensitivity. A 220-pL volume of the acid butanol phase was transferred to a 96-well plate, and the absorbance was read at 550 nm with a plate reader (Molecular Devices). Controls were as stated above. The initial amount of sialic acid attached to fetuin was measured by release with 0.1 N HC1 at 80 "C for 50 min (Schauer, 1987)followed by quantification using the thiobarbituric acid assay. The absorbance at 550 nm was calibrated to standard NeuAc (type VI from Escherichia coli) concentrations subjected to the same conditions. Fetuin concentrations are reported as moles of fetuin-bound sialic acid per liter. Determination of K m , Vm-9 and Ki. For the determination of Km and Vm, of the CHO lysate activity for 4MU-NeuAc, sialidase assays were run in triplicate as in the standard assay, except that the initial concentration of 4MU-NeuAc was varied at 0.1, 0.2,0.4, 1, and 2 mM. Because up to 40% hydrolysis occurred at the lower substrate concentrations, an integrated form of the Michaelis-Menten equation was used: P = V-t + Km In (1- P/S& where P is the amount of 4MU released (mM),SOis the initial substrate concentration (mM), and t is the reaction time (0.5 h). A plot of P vs In (1- P/&) gives a slope of K m (mM) and an intercept of V-t (mM). Inhibition constants (Ki) for the lysate were obtained by determining the apparent Km in the presence of 20 pM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (2,3-D) or 4 mM NeuAc. The Ki (mM) was calculated from the relationship, Kmapp = Km(1 + I/Ki), where I is the concentration of the inhibitor (mM), Km is the Michaelis constant without inhibitor added (mM), and Km,app (mM) is the apparent Michaelis constant with inhibitor present. Kinetic parameters of CHO lysate toward fetuin at pH 5.5 were obtained by measuring sialic acid released after a 4-h incubation with initial substrate concentrations of 0.5,0.6, 0.7,0.9, and 1mM fetuin-bound sialic acid. The pH 6.5 and 7.5 data were obtained by incubation of lysate with

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PH Figure 1. pH-activity profiie of CHO lysateglycosidaseactivity toward 4MU glycoside substrates. Glycosidase activity was measured under standard assay conditions in acetate (0)or phosphate (e) buffer. Data shown represent the average and standard deviation of duplicate determinations. fetuin (six samples each at 0.5 and 1.5 mM) for 8 h. Km and V- for fetuin were determined from LineweaverBurk plots. Propagation of the standard deviation in the determined slope and intercept for each plot was used for the error estimates reported in the tables. Km, V-, and Ki determinations for the supernatant activity toward 4MU-NeuAc were performed as above except the concentrations of 4MU-NeuAc were 0.2 and 1 mM. Test for Sialidase Inhibitors in Cell Lysate and Supernatant. Totest for sialidase inhibitors in celllysate, an aliquot containing the lysate of 9.5 X 106 cells/mL (3.1 mg/mL cellular protein) was boiled for 10min to inactivate the sialidase activity. The sialidase activity in a fresh aliquot of CHO cell lysate was measured with4MU-NeuAc at pH 6.5 in the presence and absence of boiled lysate (40 CCL of boiled lysate per 100 pL of assay volume). To test for sialidase inhibitors in cell culture supernatant, conditioned medium from confluent CHO cells was boiled and added to a fresh aliquot of CHO cell lysate as above (40pL of conditioned medium per 100pL of assay volume). Results CHO Lysate GlycosidasepH-Activity Profiles. The activities of sialidase, 8-galactosidase, 8-hexosaminidase, and fucosidasewere assessed in lysates of the WB1 DHFRCHO cell line using 4-methylumbelliferyl substrates (Figure 1). Sialidase activity was optimal at pH 5.5, with a broad shoulder of activity extending through pH 7. @-Galactosidasehad the most acidic pH optimum (pH 4)

Biotechnol. hog., 1993, Vol. 9, No. 4

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Time in Culture (days) Figure 2. Extracellular glycosidase and LDH activities in samples from two CHO perfusion cultures. Frozen CHO cell culture supernatant samples were provided by Chiron Corp. (Emeryville,CA). The perfusion rate was modulated on the basis of glucose utilization rate from approximately 0.1 to 0.5 (v/v)per day. Glycosidaseactivities were measured with 4MU glycosides under standard assay conditions at pH 7.5. Error bars representing one standard deviation in triplicate measurements of sialidase activity are obstructed by the size of the data points shown (the other glycosidase activities are from single determinations; the reproducibility of these assays is generally better than 5 7% 1. LDH activity was measured in triplicate using Sigma kit no. 340-LD. Since CHO cells from the Chiron bioreactors were not available, the relative LDH activity corresponds to the level of LDH in each sample normalized on the amount of LDH released by the lysis of 1million cells per milliliter of the WB1 DHFR- CHO cell line. The two bioreactors were coded M ( 0 ) and N (0). with little residual activity at pH 7. @-Hexosaminidase had an optimum near pH 6; however,the activity declined rapidly at higher pH. Fucosidase had the most neutral pH optimum (pH 6-51,with substantial activity above pH 7. Glycosidase Activities in CHO Cell Culture Supernatant. To determine whether glycosidase activities could accumulate in the extracellular environment of commercial bioreactors, we obtained frozen, cell-free supernatant samples from two CHO cell perfusion cultures at Chiron Corp. (Emeryville, CA). A significant amount of glycosidase activity was found in these samples when assayed with 4-methylumbelliferyl substrates at pH 7.5 (Figure 2). Lactate dehydrogenase (LDH) activity was

360

measured to estimate of the number of nonviable cells per milliliter in each perfusion culture (Figure 2). The level of glycosidase and LDH activity varied between the two reactors and within each reactor over the course of culture; this is potentially explained by the fact that the perfusion rate in each reactor was varied over the range of 0.1-0.5 (v/v) per day. Such variability in operating conditions and possible variation in sample handling at Chiron complicates further interpretation of these results. The key finding in these experiments is that the four glycosidases are present in CHO cell culture supernatant at pH 7.5 in proportions that are roughly consistent with their proportions in CHO cell lysate at that pH (Figure 1); &galactosidaseactivity is barely detectable, 8-hexosaminidase and sialidase activity are present at intermediate levels, and fucosidase activity is highest (Figure 2). Characterizationof CHO Lysate Sialidase Activity. The finding of substantial sialidase activity above pH 7 in CHO cell lysates and CHO cell culture supernatant (Figures 1and 2) was in contrast to many previous reports concerning the sialidase activities in other mammalian cell lines (Warner and O'Brien, 1979;Spaltro and Alhadeff, 1984; Schauer and Wember, 1984; Sato, 1989; Potier, 1979b). Therefore, further experiments were performed to characterize the CHO cell sialidase activity. We determined that sialidase activity toward 4MU-NeuAcwas proportional to lysate concentration and that release of 4MU was linear with time. The amount of sialidase activity in CHO cell lysates varied about 30% for lots of cells harvested on different days. The fetal calf serum used as a growth supplement for these cells was devoid of sialidase activity. In further contrast to previous literature reports concerning mammalian sialidases (Conzelmannand Sandhoff, 1987; Potier et al., 1979a; Den Tandt and Leroy, 1980; Hiraiwa et al., 1987), the CHO lysate sialidase activity was very stable. No loss in sialidase activity (measured with 4MU-NeuAc at pH 4.5, 5.5, 6.5, and 7.5) in CHO lysates was detected after 10 freeze-thaw cycles or after storage for 4 months at -20 "C. The sialidase activity in CHO cell lysates exhibited a half-life of 57 h at 37 "C (Figure 3A). Thermal inactivation followed first-order kinetics with respect to time (Figure 3A,B), followed an Arrhenius-type dependence on temperature (Figure 3C), and was not influenced by assay pH. The kinetics for hydrolysis of 4MU-NeuAc by CHO lysate sialidase is consistent with previous reports for other mammalian sialidases (Conzelmann and Sandhoff, 1987). The hydrolysis followed simple Michaelis-Menten kinetics and was competitively inhibited by free N-acetylneuraminic acid (a sialic acid) and a known sialidase inhibitor, 2,3dehydro-2-deoxy-N-acetylneuraminic acid (2,3-D)(Meindl andTuppy, 1969)(Table I). The effect of temperature on the rate of hydrolysis followed an Arrhenius-type dependence and was not strongly influenced by pH (Figure 4). Next, it was established that CHO lysate sialidase can also hydrolyze sialic acid from a glycoprotein substrate, fetuin. The Vm, for fetuin is about 4 times smaller and the K m about 4 times greater than those for 4MU-NeuAc (Table I). Fetuin-bound sialic acid proved to be a competitive inhibitor of lysate sialidase activity toward 4MU-NeuAc,withaKi of 0.56 mM at pH 5.5. This suggests that the same enzyme is acting on both 4MU-NeuAc and fetuin since the Km for fetuin (Table I) is essentially identical to the Ki for fetuin-inhibited hydrolysis of 4MUNeuAc. The K, and Vmarfor fetuin apparently represent the kinetic parameters toward the intact glycoproteinsince the addition of protease inhibitors as previously described (Miyagi and Tsuiki, 1984)did not affect our experimental

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Table I. Kinetic Characteristics of CHO Lysate Sialidase Activity from the WB1 DHFR- Cell Line Provided by Genentech. pH V., 4MU-NeuAc (normalized) V,, fetuin (normalized) K, 4MU-NeuAc (mM) K, fetuin (mM) Ki NeuAc (mM) Ki 2,3-D(rM) 4.5 5.5

0.83 0.04 0.23 f 0.06 1.00 0.17 i 0.01 6.5 0.76 0.03 0.13 f 0.01 7.5 0.59 f 0.03 0 The V , values reported are normalized on the V, 4MU-NeuAc. See Materials and Methods for details.

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Figure 4. Effect of temperature on CHO lysate sialidaseactivity. CHO lysate sialidase activity was measured in duplicate under standard assay conditions with 4MU-NeuAc at pH 4.5, 5.5,6.5, and 7.5 at 5,15,25,and 37 "C. The activity was normalized on activity at 37 "C for each pH. The apparent activation energy of hydrolysis determined from an Arrhenius-typeplot was 53.8, 59.7,61.8,and67.3kJ/molatpH4.5,5.5,6.5,and7.5,respectively.

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1/T (1IK) Figure 3. Thermal stability of CHO lysate sialidase. (A and B) CHO lysate sialidase activity remaining after preincubation at various temperatures was measured in triplicate with 4MUNeuAc under standard assay conditions at pH 4.5,5.5,6.5,and 7.5. The fraction of activity remaining was not influenced by assay pH, and the results shown here for each data point are pH-averaged. Error bars represent one standard deviation in the pH-averaged data, and many of them are obstructed by the size of the data points shown. The first-order inactivation rate constant k (h-1) was determined from the slope of a semilog plot of the fraction of remaining activity vs time (h) for each temperature. (C) Arrhenius plot of In (k) vs the reciprocal temperature 1/T (K-l) gives an apparent activation energy of 238kJ/mol and apreexponentialfactor of 1.42 X 1038. The curves in A and B represent the theoretical deactivation rate computed from the activationenergy and preexponential factor determined in C. results with fetuin as substrate (or the corresponding control using 4MU-NeuAc). The level of extracellular degradation of product glycoproteins by glycosidases could potentially be minimized if inhibitors are normally present in the culture supernatant. For example,there are many potential competitive glycoprotein substrates, such as glycoprotein medium components (e.g., transferrin), cell surface glycoproteins,

The curve shows a best fit for pH-averageddata with an apparent activation energy of 62.2 kJ/mol. and glycoproteins released from lysed cells. We found that the activity of CHO cell lysate sialidase toward 4MUNeuAc was not significantly inhibited by the addition of boiled lysate or the addition of conditioned CHO cell culture medium containing 5% fetal bovine serum (as described in Materials and Methods). This lack of significant inhibition in spite of the presence of alternative glycoprotein substrates can be explained as follows. The K, of the CHO lysate sialidase toward fetuin at pH 7.5 is quite high (Table I), corresponding to a glycoprotein concentration on the order of 7.4 g/L based on a fetuin molecular weight of 48 400 and 13.1 mol of sialic acid per mole of fetuin (Spiro, 1960). If the K, of CHO sialidase activity toward other glycoprotein substrates is of comparable magnitude, then we would not expect significant saturation (competitive inhibition) of the CHO sialidase by alternative glycoprotein substrates in cell lysates or in the cell culture Supernatant. Characterizationof CHO Cell Supernatant Sialidase Activity. The kinetic parameters for hydrolysis of 4MU-NeuAc by CHO cell supernatant sialidase were investigated for comparison to the CHO lysate sialidase activity. All of the of Chiron CHO cell supernatant samples were pooled for this study. The K m and V,, for CHO supernatant sialidase were found to be very similar to CHO lysate sialidase across the pH range of 4.5-7.5, as was the Ki for inhibition of 4MU-NeuAc activity by 2,3-D (Tables I and 11). The quantity of Chiron CHO cell culture supernatant available was insufficient to perform a kinetic analysis for release of sialic acid from a glycoprotein substrate. To determine whether CHO supernatant sialidase is capable of releasing sialic acid from a glycoprotein substrate, a partially purified supernatant sialidase preparation from CHO cell batch culture was provided by Genentech. This partially purified sialidase preparation had a V,, of 805 nmol/h/mL and a Kmof 0.48 mM toward

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Table 11. Kinetic Characteristics of Extracellular CHO Sialidase Activity from SuDernatant Provided by Chiron. ~~

V,,

4MU-NeuAc (normalized) 0.75 0.04

K,

Ki

4MU-NeuAc 2,3-D UH (mM) (PM) 0.18 f 0.03 16f 1 4.5 0.14 f 0.02 8f1 5.5 1.00 0.29 f 0.03 9f2 6.5 0.78 f 0.04 0.43 f 0.02 14 f 4 7.5 0.53 f 0.02 a The V,, values reported are normalized on the V,, for 4MUNeuAc at pH 5.5. The Ki values are for inhibition of activity toward 4MU-NeuAc. See Materials and Methods for details. 4MU-NeuAc at pH 7.5. Fetuin proved to be a substrate for this extracellular sialidase at pH 7.5, with an activity of 106 f 13 nmol/h/mL (n = 3) with fetuin-bound sialic acid at a concentration of 2 mM. These results are consistent with the previously obtained results for the sialidase activity in CHO cell lysate. That is, an activity of 91 f 9 nmol/h/mL would have been predicted for release of sialic acid from fetuin by the supernatant sialidase on the basis of the activity toward 4MU-NeuAc in the supernatant and the relative rates of sialic acid release from 4MU-NeuAc and fetuin shown in Table I for the lysate activity. These results demonstrate that CHO supernatant sialidase is capable of altering the oligosaccharide structures of glycoproteins extracellularly. Sialidase Activity in T-Flask Supernatant. Supernatant from a T-flask of the WB1 DHFR- CHO line was examined to determine whether extracellular sialidase activity was significant under conditions of relatively low cell density (9.3 X 106 cells/mL) and high cell viability (>95 % ). The extracellular sialidase activity measured with 1 mM 4MU-NeuAc was 0.3 nmol/h/mL at pH 7.5. The low level of extracellular sialidase detected under these cell culture conditions corresponds to approximately 1% of the intracellular sialidase activity associated with 9.3 X l o 5 cells/mL. Discussion This study represents the first demonstration that CHO cells possess a stable sialidase activity that can accumulate in the extracellular medium. In contrast to previous reports concerning mammalian sialidases (Warner and O’Brien, 1979; Spaltro and Alhadeff, 1984; Schauer and Wember, 1984; Sato, 1989; Potier, 1979b), the sialidase activity in CHO cell lysates retains considerable activity at pH 7 and above (Figure 1). The presence of fucosidase, 8-galactosidase, and @-hexosaminidaseactivities in the extracellular medium of CHO cell cultures has been previously demonstrated using 4-methylumbelliferyl substrates at acidic pH (Robbins, 1979; Hall et al., 1986). In this study, we present the pH-activity profiles of these glycosidases in CHO cell lysate and demonstrate their presence in CHO cell culture supernatant at pH 7.5 (Figures 1 and 2). These results suggest that the heterogeneity of glycoprotein oligosaccharides could be influenced by endogenous CHO cell sialidase released into the extracellular medium. Further evidence has recently been presented by Dr. Mary Sliwkowski and co-workers at Genentech. They produced deoxyribonuclease (DNase) by recombinant CHO cells in batch culture in the presence and absence of 2,3-D using tissue culture plates where pH was not controlled. Toward the end of the batch culture, they observed a shift in the IEF pattern of DNase produced in the absence of 2,3-D that would be consistent with the loss of sialic acid from DNase oligosaccharides. In the presence of 2,3-D, the IEF pattern at the end of the batch culture

retained a profile consistent with the retention of sialic acid on the DNase oligosaccharides (Sliwkowski et al., 1992). The magnitude of degradation by extracellular sialidase would depend upon many factors, including the level of extracellular sialidase activity, pH, temperature, and the time period of glycoprotein exposure to the sialidase. On the basis of the data presented in the Resulta section, a calculation can be performed to determine whether the level of sialidase found in the Chiron perfusion bioreactors is of an order of magnitude sufficient to remove asignificant amount of sialic acid from a secreted glycoprotein. The degradation reaction rate r (mol/L/h) = V-S/[Km(l + I/Ki) + SI,where V m a is the maximal reaction rate (mol/ L/h), S is the concentration of the glycoprotein of interest (mM), K , is the Michaelis constant (mM), I is the concentration of other possible competitive inhibitors (mM),andKiis the inhibition constant of the other possible competitive inhibitors (mM). On the basis of considerations outlined in the Results section, it is probably reasonable to assume that the concentration of the secreted glycoprotein substrate will always be less than Km and that competitive inhibition by other glycoproteins in the supernatant will be insignificant. Therefore, the equation further reduces to the first-order reaction rate r = (VmJ Km)S. On this basis, an estimated 10% of the glycoproteinbound sialic acid would be released by sialidase in the Chiron CHO perfusion bioreactor M in Figure 2, while removal of 26 % of the sialic acid is estimated in bioreactor N. These estimates were calculated by assuming the average activity in each reactor, a constant perfusion rate of 0.25 (v/v) per day, a constant glycoprotein secretion rate, and the kinetic parameters for fetuin relative to 4MUNeuAc at pH 7.5 in Table I. Release of sialidase into the extracellular medium by CHO cells could potentially be due to cell lysis and/or secretion due to intracellular missorting. The data presented in this study were insufficient to draw any specific conclusions regarding these release mechanisms. However, the recognition of such mechanisms does suggestthat there could be considerable variability in the extracellular concentration of sialidase, depending upon cell culture conditions-that is, extracellular hydrolysis of glycoprotein oligosaccharides by sialidase could be insignificant under conditions which minimize extracellular sialidase accumulation. For example, the supernatant from CHO cells grown at low density and high viability in T-flasks contained little sialidase activity (0.3 nmol/h/mL at pH 7.5) after a 2-day incubation period. Had this cell line been secreting a glycoprotein, extracellular degradation by sialidase would have been insignificant under these conditions. Differential exposure to extracellular glycosidasescould explain, a t least in part, why the same recombinant glycoprotein produced in CHO cell culture by different groups can have strikingly different oligosaccharide structures-for example, the oligosaccharide structures of t-PA produced by Monsanto and analyzed by the Oxford University group (Parekh et al., 1989) versus t-PA produced and analyzed by Genentech (Spellman et al., 1989) and soluble CD4 produced by Invitron and analyzed at SmithKline (Yuen et al., 1990)versus CD4 produced and analyzed by Genentech (Spellman et al., 1991). For t-PA and CD4, the oligosaccharide structures of the glycoproteins produced by Monsanto and Invitron show decreased monosaccharide content (less sialic acid, less galactose, less fucose, etc.) relative to the same proteins produced at Genentech. Some of these deficiencies could be attributable to differences in biosynthesis, purification protocol, or even the method of oligosaccharide analysis. However,

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some of the oligosaccharide structures of the Invitronproduced CD4, including a monoantennary structure (see fraction 11-E in Figure 5 of Yuen et al. (1990)),are difficult to explain on the basis of any known biosynthetic route (reviewed in Kornfeld and Kornfeld (1985)), suggesting that extracellular degradation by glycosidases may have occurred in this case. A key point from our analysis of the Chiron and Genentech bioreactor supernatants is that considerable sialidase activity can accumulate in the extracellular environment. The amount of sialidase activity found in the Chiron and Genentech supernatant samples would be released from just 2 X lo5 to 5 X lo5 lysed cells/mL, a relatively small number of lysed cells given the cell densities that are currently achievable-that is, this would represent release of sialidase from just 2 to 5% of cells at a total cell density of 1X lo7cells/mL. The industrial trends toward higher cell densities and maintenance of high-density cultures for longer time periods provide strong motivation for further study of the potential for extracellular release of sialic acid from glycoprotein oligosaccharides by CHO sialidase.

Acknowledgment We thank Dr. Mary SIiwkowski (Genentech) and Dr. Peter Brown (Chiron) for providing CHO cells and CHO cell culture supernatant for this study. In addition, we thank Dr. Sliwkowskifor her helpful comments concerning this project. This work was supported through a National Science Foundation Presidential Young Investigator Award to C.G. and a Department of Defense fellowship to M.G. These results were presented a t the Annual Meeting of the American Institute of Chemical Engineers, Los Angeles, CA, November 1991.

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Accepted March 19, 1993.